Purpose :
Soldiers are faced with a variety of ocular injuries, often leading to the need for corneal transplant. Throughout their lifetime, the average Soldier will need 2-3 transplants, due to the degradation of the corneal endothelium upon transplantation or trauma. Currently, the only way to replace this layer is with a successive tissue transplant. We have previously proposed the use of magnetized human corneal endothelial cells as an alternative and less invasive therapy for this injury type. Herein, we describe an injury model to damage the endothelium in order to evaluate the effectiveness of our therapy.

Methods :
Porcine ocular tissue was collected within one hour of sacking to maintain endothelial cell integrity. Eyes were soaked in sterile PBS with 1X Anti/Anti for 20 minutes before experimental procedures. Pictures were taken of the ocular surface and the endothelial surface with counts of the density, size, and polymorphism recorded via a specular microscope. Dry Ice or 9mm cryoprobe dipped in liquid nitrogen were then used to injure the endothelium by placing on top of the cornea for up to 25 seconds. These eyes were then evaluated with relevant photography and stained for live/dead to show the occurrence of cellular death. The tissue was then fixed in Davidson’s fixative and evaluated for endothelial integrity.

Results :
On average 30% cell death occurred across both methods of corneal injury. Live/dead staining showed evidence of endothelium detachment with small clusters of cells remaining intact and viable. The cryo-probe technique created a more opaque defect upon initial contact, but the epithelium of respective eyes cleared comparably between groups. While live/dead revealed a similar percentage of viable cells between groups, imaging with the specular microscope for endothelial cell counts was more viable in the dry ice group.

Conclusions :
Transcorneal freezing presents a method of corneal endothelial damage that produces a consistent live/dead concentration for evaluation of potential therapeutics. This damage rate is retained across dry ice and the use of a cryo-probe. The damage produced herein, will be evaluated in an animal model and used as a platform to evaluate effectiveness of cellular therapeutics.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.